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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2689-2697
Chronic Lymphocytic Leukemia B Cells Can Express CD40
Ligand and Demonstrate T-Cell Type Costimulatory Capacity
By
Elaine J. Schattner,
John Mascarenhas,
Inna Reyfman,
Mary Koshy,
Caroline Woo,
Steven M. Friedman, and
Mary K. Crow
From the Division of Hematology-Oncology, Department of Medicine, The
New York Hospital-Cornell Medical Center, New York, NY; and the
Division of Rheumatology, The Hospital for Special Surgery, New York,
NY.
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ABSTRACT |
Chronic lymphocytic leukemia (CLL) is characterized by a clonal
expansion of CD5+ B cells in the peripheral blood.
Associated immune aberrations include abnormal Th-cell
function and pathogenic autoantibodies. Under most circumstances, CLL B
cells do not proliferate in culture and express a limited repertoire of
surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In
this report, we demonstrate that freshly isolated B cells from a subset
of CLL cases constitutively express CD40 ligand (CD40L, CD154), a
member of the tumor necrosis factor family which is normally expressed
by activated CD4+ T cells and mediates T-cell-dependent
B-cell proliferation and antibody production. The degree of CD40L
expression varied considerably among the CLL cases examined. CD40L was
detected in purified CLL B cells by immunofluorescence flow cytometry,
by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the
CLL B cells is functional, we used irradiated CLL cells to stimulate
IgG production by target, nonmalignant B cells in coculture. The CLL B
cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by
monoclonal antibody to CD40L. This is one of the first reports of CD40L
expression in a B-cell tumor. The data suggest that CD40L in the tumor
cells may be a factor in the generation of pathologic antibodies by
normal B cells in some patients with CLL.
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INTRODUCTION |
CHRONIC LYMPHOCYTIC leukemia (CLL) is the
most frequent adult leukemia in the United States and is usually noted
due to an excess of well-differentiated B lymphocytes in the peripheral blood. The diagnosis is confirmed by fluorescence flow cytometry of the
lymphocytes, which characteristically express cell surface antigens
CD5, CD19, and CD23.1 Although the prognosis for early stage CLL is excellent, many patients with only a lymphocytosis require
treatment for and suffer complications from immune manifestations of
this disease.2-4 Autoimmune sequelae of CLL include both
Coomb's positive hemolytic anemia and thrombocytopenia.5,6
Independent of treatment, patients with CLL are vulnerable to
infectious pathogens due to impaired humoral immunity characterized by
hypogammaglobulinemia.7-10 A variety of T-cell derangements
have also been observed, which include a T-cell lymphocytosis, an
inverted ratio of CD4+ to CD8+ T
cells,11 deficient T-cell help,12,13 and
reduced capacity to secrete cytokines such as interleukin-2
(IL-2).14
CD40 ligand (CD40L; CD154, gp39, T-BAM)15 is
mainly expressed in activated, CD4+ T cells and, along with
CD28, is one of the main costimulatory signals by which specific,
antigen-reactive T cells offer help to B cells presenting that antigen
in the context of MHC-II.16-19 The receptor on the B cell
for CD40L is CD40, a member of the tumor necrosis factor (TNF) receptor
superfamily that is expressed in almost all B lymphocytes as well as in
macrophage and endothelial cells.20 Ligation of CD40 by its
ligand, CD40L, in the germinal center B cell results in inhibition of
apoptosis,21 proliferation, differentiation with Ig isotype
switching,22-24 and expression of activation antigens,
including CD2317,25 and Fas.26,27 As in most
B-cell tumors, CLL B cells express CD40,28-31 and several groups have observed that CLL B cells can be induced to differentiate into Ig-secreting cells.32-34
Normal T lymphocytes express CD40L for only a few hours after
activation. In T cells derived from patients with systemic lupus erythematosus (SLE), CD40L expression is increased and
prolonged.35,36 Several groups have described CD40L
expression in pathologic samples from certain T-cell malignancies and
also in human T-cell line cells.37,38 Whether human B cells
express CD40L is less established. Grammer et al39
demonstrated that human peripheral blood B cells could be induced to
express CD40L subsequent to stimulation with calcium ionophore and
phorbol ester, and that Epstein-Barr virus (EBV)-transformed
lymphoblastoid cell line cells (LCLs) express functional CD40L after
exposure to B-cell mitogens.39 Additional reports indicate
that CD40L is expressed in certain human B-cell tumors40
and in B cells from patients with SLE.36
In this work, we analyzed freshly purified CLL B cells and found that,
in a subset of CLL patient samples, there was significant CD40L
expression in the unstimulated malignant cells. CD40L in the CLL B
cells was functional in a costimulatory capacity, in that the CLL cells
induced antibody production by nonmalignant, human B cells. These
results provide evidence for a possible mechanism of paracrine tumor
stimulation in CLL and for excess autoantibody production due to
inappropriate B-cell "help."
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MATERIALS AND METHODS |
Patient samples.
Approval for the research protocol was obtained from the internal
review boards of The New York Hospital and The Hospital for Special
Surgery, and verbal, informed consent was obtained before phlebotomy.
In each case, the diagnosis of CLL was established by the coexpression
of CD5 and CD19 in an expanded, clonal population of peripheral blood
lymphocytes. Mononuclear cells were isolated from 10 to 30 mL of fresh,
heparinized peripheral blood by Ficoll-Hypaque centrifugation. For most
of the experiments, T and B lymphocytes were separated by rosetting
with sheep red blood cells according to standard techniques, and the
degree of separation was checked by immunofluorescence flow
cytometry.
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| Fig 1.
CD40L expression in freshly purified CLL B cells. (A)
Peripheral blood mononuclear cells from patients with CLL (all
untreated for at least 4 months) were purified by Ficoll-hypaque
centrifugation and depleted of T cells by rosetting with sheep red
blood cells. Shown are the results of immunofluorescence flow cytometry
in consecutive, unselected cases for which the cells could be stained immediately after purification. In each case, greater than 96% of the
cells expressed high levels of CD19, and in the majority greater than
99% of the purified cells expressed CD19. As shown, each histogram
except for the first represents the fluorescence intensity after
incubation with an irrelevant, isotype-matched control antibody (murine
IgG1 antihuman TcRV 13, shaded histograms) or
with anti-CD40L (open histograms), followed by goat antimouse-FITC. In
the first case, the shaded histogram indicates CD3 expression. (B)
Peripheral blood mononuclear cells were washed briefly in neutral (pH
7.1) or acidic PBS (pH 4.1) to remove soluble CD40 from the
surface39 before exposure to MoAbs for immunofluorescence cytometry. After washing in neutral PBS, cells from each sample were
examined with CD19-FITC and CD3-FITC concomitantly with either CD40L-PE
(clone 89-76) or with a negative, control PE-conjugated antibody. In
each plot, the linear histogram reflects PE fluorescence intensity
after exposure to CD40L-PE and the shaded histogram reflects
fluorescence intensity after exposure to the control antibody, in each
case analyzed among the gated, CD19-FITC-positive cells.
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| Fig 2.
Two-color immunofluorescence demonstrates B-cell
expression of CD40L. (A) Unfractionated, peripheral blood cells
from patients were analyzed by two-color immunofluorescence flow
cytometry immediately after isolation. Shown are three examples
including the best case (no. 10), an intermediate case (no. 25), and a
low CD40L (CD154) case (no. 28). Each contour plot reflects expression
of CD19 on the x-axis, demonstrated with a directly conjugated
anti-CD19-FITC MoAb. The plots on the left demonstrate the results
after exposure also to a PE-conjugated antibody to CD5, those in the
center after exposure to a PE-conjugated antibody to CD3, and those on
the right after exposure to a PE-conjugated antibody to CD40L (TRAP clone). The percentage of double-positive cells for each sample is
indicated.
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Cell culture.
Unless otherwise indicated, cells were analyzed immediately subsequent
to their purification. For some experiments, as indicated, cells were
cultured in C50 media (RPMI supplemented with penicillin-streptomycin with L-glutamine and 10% fetal calf serum [FCS]) only or with IL-2
at 40 U/mL (Hemagen Diagnostics, Waltham, MA) and a 1:60,000 dilution
of formalinized Staphylococcus Aureus Cowan 1 (SAC; Pansorbin; Calbiochem, San Diego, CA), recombinant IL-4 at 10 ng/mL (Genzyme Diagnostics, Cambridge, MA), monoclonal antibody (MoAb) to CD40 at 1 µg/mL (IgG 1; Genzyme), an F(ab )2
preparation of MoAb to human IgM at 0.5 µg/mL (Jackson Immunoresearch
Laboratories Inc, West Grove, PA), phorbol myristate acetate (PMA) at
10 ng/mL (Sigma) and ionomycin at 1.25 µg/mL (Calbiochem), or
pokeweed mitogen (PWM; Life Technologies, Gaithersburg, MD). For cDNA
preparation, Jurkat mutants that constitutively express CD40L (clone
D1.1) were used as a source of CD40L.41 These mutant Jurkat
cell lines were the kind gift of Dr Seth Lederman (Columbia University,
New York, NY). The Ramos Burkitt's lymphoma cell line was obtained from American Type Culture Collection (ATCC; Rockville, MD). For immunoprecipitation experiments, in addition to the CLL samples, we
used purified CD4+ polyclonal T-cell lines derived from
patients with B-CLL that respond to the toxic shock syndrome toxin-1
(TSST-1; Toxin Technology, Sarasota, FL) superantigen. These cells were
maintained in culture with IL-2 at 40 U/mL and were periodically
triggered with TSST-1 (used at 10 ng/mL) in the presence of allogeneic
antigen-presenting cells, as described previously.27,42
Flow cytometry and immunofluorescence analysis.
Cells were washed in cold Hank's Buffered Saline Solution (HBSS) and
incubated with antibodies according to standard techniques. Cell
fluorescence was measured using a Becton Dickinson FACScan (Becton
Dickinson, San Jose, CA). Cells were assessed for forward and side
scatter, and events in the viable cell gate were analyzed for
fluorescence intensity using the CellQuest program (Becton Dickinson).
For acid washing of cells before analysis, the cells were exposed to
acidic PBS (pH 4.1) for 3 minutes at room temperature and then washed
twice with normal pH before exposure to MoAbs, as indicated, similar to
the technique reported by Grammer et al.39
MoAbs.
Sterile antibodies used in cell culture experiments included anti-CD40
(murine IgG1; Genzyme), anti-CD40L (murine
IgG1,, clone M-90; Genzyme), and anti-CD23 (EBVCS2; ATCC).
In the blocking coculture experiments, the dose of each antibody used
was 5 µg/mL. For single-color fluorescence flow cytometry, the
antibodies used were anti-CD3 (OKT3; ATCC), anti-V 13 (murine
IgG 1), anti-CD19 (Immunotech, Westbrook, ME), anti-CD40 (Genzyme),
and anti-CD40L (clone M-90, murine IgG1; Genzyme), in each case
followed by goat antimouse Ig-fluorescein isothiocyanate (FITC).
Antibodies used for two-color analyses included anti-CD3-FITC
(Immunotech), anti-CD40-FITC (Biosource, Camarillo, CA), anti-CD19-FITC
(Immunotech), anti-CD3-PE (Biosource), anti-CD5PE (Biosource),
anti-CD40L-PE (TRAP clone; PharMingen, San Diego, CA), and
anti-CD40L-PE (clone 89-76; Becton Dickinson).
Reverse transcriptase-polymerase chain reaction (RT-PCR).
RNAs were isolated from approximately 1 × 107 cells
using the TRIzol reagent (Life Technologies/GIBCO BRL, Grand Island,
NY), according to the manufacturer's instructions, and
the yield in each case was measured by spectroscopy. For each sample,
cDNAs were prepared using approximately 1 µg of RNA, using a reverse transcriptase kit (GIBCO). For CD40L, the primers used were
5-GGCCATTATGCACAGGTTGAAT-3 (1100-1122) and
5-GGGGAGGGAAGAGACTGACAAA-3 (1469-1489),43 which yielded a single amplified CD40L fragment of 396 bp. The quality
of each cDNA preparation was checked by amplification of GAPDH
sequences using primers 5-GAAGATGGTATCACCTGGAC-3 and 5-GAAGAAGCTGCTGGTGTAGT-3 (Stratagene, La Jolla, CA),
which yield a single, amplified fragment size of 600 bp.
Immunoprecipitation.
Freshly isolated purified CLL B cells were placed in culture under a
variety of circumstances. After 36 or 60 hours as indicated, the cells
were washed and cell surface proteins biotinylated using Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL).44 Lysates were
then subjected to immunoprecipitation overnight at 4°C using 3 µg
of antibody to CD40L (TRAP clone; PharMingen) or with 3 µg of a
control, isotype-matched antibody to IL-4 (Genzyme), in each case
conjugated to protein A Sepharose 4 Fast Flow beads (Pharmacia Biotech,
Piscataway, NJ). Precipitated materials were boiled for 4 minutes before sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride
(PVDF) membrane, exposed to horseradish peroxidase
(HRP)-conjugated streptavidin (Pierce) for 90 minutes, and
then imaged using a chemiluminescence substrate (Amersham,
Buckinghamshire, UK).
Ig measurement.
IgG and IgA secreted into the supernatants was measured by a standard
enzyme-linked immunoabsorbance assay (ELISA) protocol. In each
experiment, standard curves for the particular isotype were determined,
and the samples were diluted such that the absorbance readings fell in
the linear range of detection. For each circumstance and in each
experiment, antibody measurements were determined in triplicate.
Results were discounted if the standard deviation among the three
triplicate readings represented greater than 15% of the mean or if the
result for the diluted sample did not fall within the linear range of
the standard curve. IgG production, as shown in Fig 6, reflects the
results of single-case analyses, with error bars indicating the
standard error for measurements of each experimental circumstance.
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| Fig 6.
CLL B cells offer T-cell costimulatory type help to
target, nonmalignant B cells. (A) CLL B cells induce normal, target B cells to produce antibodies in the presence of PWM. A total of 5 × 104 autologous T cells (AutoTxr) from the
normal target B-cell donor, CLL B cells (CLLBxr), or T
cells derived from the CLL patient (CLLTxr) were irradiated
(2,000 rad) and placed in culture with 5 × 104 target,
purified B cells from the normal donor, in the absence or presence of
PWM. Control circumstances included 5 × 104 target,
unirradiated B cells alone, with and without PWM, and 5 × 104 irradiated CLL B cells, in the absence of target B
cells. IgG production was assayed by ELISA from supernatants obtained
after 7 days of coculture. (B) CLL B-cell-induced, PWM-dependent IgG production by normal B cells is inhibited by antibody to CD40L. The
experiment shown is typical of a series in which 8 × 104
irradiated CLL B cells were exposed to MoAbs (final concentration, 5 µg/mL) before coculture with 4 × 104 normal,
unirradiated B cells and assayed for IgG production. In this case, CLL
B cells from case no. 13 induced greater than 400 ng/mL of IgG in the
supernatant at 7 days, in an effect that was reduced in the presence of
antibodies including antibody to CD40L.
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RESULTS |
CD40L expression is evident in some CLL B cells by immunofluorescence
flow cytometry.
CLL B cells from patients with untreated CLL were purified from
heparinized peripheral blood samples by Ficoll hypaque centrifugation and separated from the T lymphocytes by rosetting with sheep red blood
cells. Of the non-T cells obtained, greater than 96% of the cells in
each case expressed CD19. In the majority of cases, greater than 99%
of the purified cells were B cells. These cells were analyzed directly,
without stimulation, by immunofluorescence flow cytometry for B- and
T-cell antigens, including CD40L. In the first series
(Fig 1A), B cells from consecutive, unselected cases
were purified and examined using an MoAb to CD40L (clone M-90) or, as a
negative control, an isotype-matched control antibody to a T-cell
receptor (TCR) chain (anti-V13). As shown, the level of CD40L
expression varied considerably among the CLL samples but significant
antibody binding was evident in 6 (cases no. 1, 4, 10, 11, 13, and 18)
of these first 13 cases analyzed.
To demonstrate definitively that the CD40L signal was not due to a
small proportion of T cells in the samples, two-color
immunofluorescence flow cytometry was performed using a directly
conjugated fluorescent antibody (TRAP clone) on additional unselected
samples, also immediately after purification of the cells. As shown in
Fig 2, in some cases, the CD19+ (B) cells
demonstrated CD40L expression above background, as determined with a
PE-conjugated antibody to CD3. Using this technique, the molecule was
detected in approximately one fourth of the cases analyzed. In 1 case
(no. 10) for which we had demonstrated fairly strong expression in
initial analyses (Fig 1), we were able again to obtain fresh cells 6 months later. This two-color immunofluorescence pattern in the upper
panel of Fig 2 represents the best example of the two-color stains we
have performed. More typical were intermediate results, as shown in the
middle panel of Fig 2 (6 cases), or low-level expression, as in the
bottom panel (15 cases). Of note, in two-color FACS analyses of greater
than 20 fresh specimens from patients with CLL, we have never observed
significant CD40L expression in the unstimulated T
cells.
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| Fig 3.
Modulation of CD40L expression in CLL B cells by
stimulation in vitro. Freshly isolated CLL B cells were exposed to
media alone (A); to the combination of IL-2 (40 U/mL) and SAC (1:60,000 dilution) (B); to IL-4 (10 ng/mL) (C); to a murine MoAb to CD40 (1 µg/mL) (D); or to an F(ab)2 preparation of
antihuman IgM (0.5 µg/mL) (E). After 60 hours in culture, the cells
were washed and then analyzed by two-color immunofluorescence cytometry
using CD19-FITC and CD3-FITC, together with CD40L-PE (clone 89-76) or with a negative, control PE-conjugated antibody. The linear histograms reflect PE fluorescence intensity after exposure to CD40L-PE and the
shaded, background histograms reflect PE fluorescence intensity after
exposure to a control antibody, in each case analyzed among the gated,
CD19-FITC-positive cells.
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| Fig 4.
CD40L message is detected in RNAs prepared from CLL
samples. Fresh CLL B cells (1 × 107) purified from the
peripheral blood of 2 patients were exposed overnight in culture to
media alone, to SAC and IL-2 (1:60,000 and 40 U/mL), to PMA and
ionomycin (10 ng/mL and 1.25 µg/mL), or to IL-4 (10 ng/mL). After 18 hours, RNAs were isolated, cDNAs were prepared by reverse
transcriptase, and CD40L sequences were amplified (A). The RNAs
analyzed are as follows: lane 1, CLL case no. 15 B cells with media;
lane 2, with IL-2 and SAC; lane 3, with PMA and ionomycin; lane 4, with
IL-4; lane 5, CLL case no. 16 B cells with media; lane 6, with IL-2 and
SAC; lane 7, with PMA and ionomycin; lane 8, with IL-4. Positive and
negative controls for detection of CD40L RNA sequences included RNAs
from 1 × 107 D1.1 cells (lane 10, Jurkat mutants that
constitutively express CD40L) and Ramos Burkitt's lymphoma B cells
(lane 9, these do not express CD40L). The cDNA preparations were
evaluated by amplification of GAPDH sequences (B).
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| Fig 5.
Immunoprecipitation of CLL B-cell lysates with MoAb to
CD40L. (A) CLL B cells were cultured with media alone (lanes 1 and 2)
or IL-4 at 10 ng/mL (lanes 3 and 4) and, after 36 hours, cell surface
proteins were biotinylated and lysates were prepared. For each
circumstance, lysates from 6 × 106 cells were subjected
to immunoprecipitation using MoAb to CD40L (TRAP clone, lanes 1 and 3)
or MoAb to IL-4 (lanes 2 and 4). As shown, the precipitated monoclonal
Ig heavy chain (46 kD, designated "H") and light chains (~29
kD, designated "L1" or "L2" for the chains of the antibody to CD40L and to IL-4, respectively) are evident in addition to the CD40L-specific bands. The 39- and 32-kD bands, corresponding to the two transmembrane forms of CD40L, are
indicated with thick arrows, and the soluble forms of CD40L, which
include a doublet at 18 kD and an additional, single band at 15 kD, are
indicated with narrow arrows. (B) CLL B cells were stimulated for 60 hours in culture with IL-2 and SAC, after which cell surface proteins
were biotinylated and lysates prepared. In this experiment, 210 µg of
protein were used in each immunoprecipitation, either with MoAb to
CD40L (lane 1, TRAP clone) or to IL-4 (lane 2). (C) Lysates from
cultured, superantigen-reactive, CD4+ T cells derived
from a CLL patient were used for immunoprecipitation of CD40L after
biotinylation of cell surface proteins. Lane 1 shows a 39-kD band after
lysates from 1 × 106 T cells were used for
immunoprecipitation. In lanes 2 (5 × 105 T cells) and 3 (2.5 × 105 T cells), the band is not evident.
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| Fig 7.
Model for CLL B-cell tumor growth and antibody production
by nonmalignant B cells due to aberrant expression of CD40L in
CD40+ CLL B cells. (A) Autoligation of CD40 by CD40L at
the CLL B-cell surface, or possibly secreted by the same cell, results
in intracellular tumor cell signaling. (B) Ligation of CD40 in one CLL
B cell by CD40L in a nearby CLL cell results in tumor cell activation,
proliferation, and inhibition of apoptosis. This process is likely to
depend also on cytokines derived from nearby T lymphocytes. (C)
Ligation of CD40 in a bystander B cell by CLL B-cell-derived CD40L
results in activation, differentiation, and antibody production by the nonmalignant cell. As in (B), this process is likely to depend on
cytokines derived from nearby T lymphocytes, and on the presence of
antigen.
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Because there is some evidence that detection of CD40L by
immunofluorescence flow cytometry may be dependent on the particular antibody used and that recognition of CD40L at the cell surface may be
hindered by the presence of soluble CD40,45 we elected to
evaluate CD40L in CLL B cells using an additional MoAb (clone 89-76).
In addition, we examined the effect of acid-washing CLL B cells with
PBS (pH 4.1) before exposure to MoAb to CD40L for FACS analysis,
according to the technique of Grammar et al,39 to remove
soluble CD40 that may be adherent to the B cell, particularly as may
occur in CD40-expressing B-cell tumors such as
CLL.45 Using the 89-76 clone, 3 of 6 cases
analyzed were positive, including 2 that were also positive with the
TRAP clone. The effect of acid-washing was similar to that reported by
Grammer et al39 using lymphoblastoid B cells and is shown
in Fig 1B.
CLL B-cell expression of CD40L is impacted by stimulation in vitro.
In cases that were positive, CD40L expression diminished rapidly over
time in culture with media only, such that the signal was less intense
48 hours after isolating the cells, became further diminished after 72 hours, and was undetectable thereafter (data not shown). However, some
stimuli did appear to augment CD40L in cases that had initially
undetectable levels. Figure 3 shows CD40L expression in
CD19+ cells from a single case that were treated for 48 hours after purification under a variety of experimental circumstances.
As shown in Fig 3B, the combination of IL-2 and SAC in culture was associated with increased CD40L expression in the CLL B cells. This
effect was observed in each of 5 cases for which we analyzed this
effect. In this particular sample, IL-4 did not result in augmented
CD40L expression (Fig 3C), but the effect of this cytokine has varied
among the 6 cases analyzed so far. Other B-cell stimulants also appear
to augment CD40L expression in the CLL cells. For example, signaling
induced by MoAb to CD40 (Fig 3D) and through ligation of the B-cell
surface antigen receptor (Fig 3E) both resulted in increased CD40L
expression at the cell surface.
CD40L message is readily detected in purified B cells from CLL
patients.
B cells were purified from 2 clinical CLL samples and placed in culture
for 16 hours before RNA extraction for analysis by RT-PCR. As shown in
Fig 4, CD40L message was readily detected in both of the
CLL samples exposed in vitro to media alone (lanes 1 and 5). In
addition, there was an increase in message in cells from the second
case after exposure to the combination of IL-2 and SAC (lane 6), to PMA
and ionomycin (lane 7), and to IL-4 (lanes 8), as compared with the
amplified signal for the housekeeping gene GAPDH. These results
correlated well with those obtained by immunofluorescence flow
cytometry.
CD40L can be detected in CLL samples by immunoprecipitation.
MoAb (TRAP clone) was used to precipitate CD40L from several samples.
For these experiments, lysates were prepared after biotinylation of
cell surface proteins. Material from 6 × 106 cells
was precipitated with either antibody to CD40L or with an
isotype-matched control antibody to IL-4 (Fig 5A). As
shown, we observed major bands at 39 and 32 kD corresponding to the
transmembrane forms of CD40L,46,47 which in this case were
most evident after exposure of the cells to IL-4 (lane 3). In addition,
a doublet was evident at 18 kD and another, single band at 15kD, as
have been reported48 and attributed to soluble
CD40L.49 The CD40L bands were not seen after
immunoprecipitation with a control antibody to human IL-4 (lanes 2 and
4). In a separate experiment, to control for the amount of protein in
each sample rather than cell number, CLL cells were stimulated for 60 hours with IL-2 and SAC and then examined for CD40L using 210 µg of
biotinylated protein in each sample (Fig 5B). As shown, there is a
prominent 39-kD band apparent in the stimulated cell lysate
precipitated with antibody to CD40L, but not with the control antibody.
Finally, to determine that the CD40L signal was not due to a small
fraction of T cells in each sample, we used activated polyclonal CD4+ T cells, derived from a CLL patient and triggered
periodically with TSST-1 and IL-2, for immunoprecipitation after
biotinylation according to the same technique. These CLL
patient-derived T cells bear an activated phenotype and express
CD40L.49a As shown in Fig 5C, CD40L was not
detected by immunoprecipitation in our system when 5 or 2.5 × 105 biotinylated T cells were used (lanes 2 and 3), but was
readily observed after 1 × 106 T cells were used
(lane 1). Thus, the absolute minimum number of T cells required to
provide the signal we observed in the CLL samples would be greater than
8% of the 6 × 106 cells used in each
immunoprecipitation, which was not the case. In the cells used for the
experiment in Fig 5A, the proportion of B cells (CD19+,
CD3 ) was 97% after culture with media alone and
98% after exposure to IL-4. In Fig 5B, the proportion of B cells was
greater than 99.5% at the time of lysate preparation.
CLL B cells do not proliferate spontaneously in vitro.
Other investigators have determined that ligation of CD40 results in
CLL B-cell activation31,49 and proliferation.50 In our laboratory, we performed thymidine incorporation assays to
examine CLL B-cell proliferation in culture under a variety of
circumstances. Given that the CD40/CD40L molecular pair is an important
modulator of CLL B cell growth, one might anticipate that the CD40,
CD40L-expressing tumor cells would proliferate spontaneously in
culture. However, as has been observed by other investigators, we did
not observe significant thymidine incorporation in any of the 6 cases
we studied without the addition of irradiated, CD40L+ T
cells (data not shown). These negative results suggest that other
factors besides CD40L, such as additional signals provided by accessory
molecules or T-cell-derived cytokines, are necessary for B-cell
proliferation in the context of CD40 ligation.
Capacity of CLL B cells to offer help via CD40L.
Because CD40L has a central role in T-cell-induced B-cell
differentiation and antibody formation, we investigated the capacity of
CLL B cells to trigger antibody production by normal, bystander B cells
in coculture. For these experiments, we used as targets B cells
purified from the peripheral blood of healthy volunteers. Effector
cells, used to stimulate antibody production by the target B cells,
included CLL B cells, autologous T cells obtained from the normal donor
in each experiment, and also T cells purified from the CLL patients.
These effector cells were irradiated and then placed in culture with
the target B cells as indicated in Fig 6. Supernatants
were removed and analyzed for IgG and IgA secretion by ELISA.
The CLL B cells were variable in their capacity to stimulate antibody
production, but in general the effects were most clear in the presence
of B-cell mitogens such as PWM. As shown in Fig 6A, the unirradiated
target B cells alone did not generate significant levels of IgG, even
with the addition of PWM. This indicates that the IgG produced was not
simply due to a few contaminating T cells in the target B-cell
preparation. In addition, the irradiated, effector CLL B cells alone
did not produce IgG in the presence of PWM. However, when irradiated
effector cells were added to the target B cells, the degree of help
provided by the CLL B cells was of the same order of magnitude as that
provided by purified, autologous T cells. Although it is possible that
some contaminating tumor-derived T cells were present among the
irradiated, effector CLL B cells, those would be unlikely to account
for a signal so strong as that obtained with 100% T cells derived from
the patient's blood. Therefore, it is highly unlikely that this result
is due to a small percentage of CLL patient-derived T cells that may have been present in the effector B-cell population.
Figure 6B shows the effects of a blocking antibody to CD40L (clone
M-90) in this coculture system. Here, the irradiated CLL B cells were
exposed either to antibody to CD40L, to CD40, or to CD23 before
coculture with the target B cells. As shown, each of the antibodies was
associated with reduced IgG production, but the result was most marked
with antibody to CD40L. Similar assays for IgA demonstrated only low
level secretion of IgA (on the order of 100 to 150 ng/mL), even in the
presence of PWM (data not shown). This result is consistent with a
complex, redundant system in which multiple factors, including CD40L,
instigate antibody production and isotype switching in B cells. Our
data demonstrate that under some circumstances, costimulatory function
and isotype switching, normally induced by T cells, can be mediated by
malignant CLL B lymphocytes.
| |
DISCUSSION |
CD40L is primarily expressed in activated T cells and has not been
considered a regular feature of any B-cell tumor. We have observed that
freshly purified B cells from a significant proportion of CLL cases
express CD40L and can induce antibody production by target,
nonmalignant B cells in a process that is partially blocked by antibody
to CD40L. Although previous investigators have established that the
CD40/CD40L molecular pair is an important modulator of CLL B-cell
growth,28,49,50 we and other investigators have observed
that CLL B cells do not proliferate spontaneously in
vitro.49-52 Therefore, the extent to which CLL
B-cell-derived CD40L impacts tumor growth may depend on costimulation
via accessory molecules and the presence of T-lymphocyte-derived
cytokines, such as IL-4.50 Taken together, the data and the
literature suggest a model for CLL tumor growth and associated immune
aberrations due to tumor cells that, at least in some cases, express
both CD40 and CD40L (Fig 7).
In work quite similar to our findings, Nusslein et al53
demonstrated that irradiated or even formaldehyde-fixed CLL B cells have the capacity to stimulate IgE production by nonmalignant B cells
in the presence of IL-4. However, our data contrast with those of
Brugnoni et al,54 who stained non-T cells from the peripheral blood of 11 CLL patients and did not detect CD40L expression in those cells. It is possible that the negative result found by
Brugnoni et al54 was due to the heterogeneity of CD40L
expression in CLL and its subtle expression in most cases. In addition,
those investigators used only the TRAP clone for CD40L, which in our laboratory offered a positive signal in only one fourth of the cases.
Closer to our work, Cerutti et al55 analyzed human B-1 (CD5+) B cells and noted above-background CD40L expression,
at levels similar to what we have observed in the human CLL cells. Most recently, while this report was under review, Trentin et
al56 described CD40L expression in some CLL cases by FACS
analysis and by RT-PCR. In this report, we include data obtained using three distinct MoAbs and, in addition, demonstrate that the signal for
CD40L in the B cells can be modulated by acid-washing the cells. Thus,
the results may depend on the particular reagent used and also on the
presence of soluble CD40 in the sample.
Ranheim et al57 reported that CLL B cells express both CD27
and its ligand, CD70, and that expression of these molecules in the CLL
cells is affected by ligation of CD40. Thus, although CLL cells are
considered as resting B cells, unable to proliferate in vitro without
T-cell-derived signals, there may be a dynamic aspect of the tumor in
vivo due to infiltrating CD4+ T cells that provide
cytokines and antigen-dependent costimulatory signals. Based on the
observations that CD40 ligation results in NF-B
activation58 and that constitutive, high NF-B activity is observed in 293 cells expressing both CD40 and CD40L,59
even low-level, transient expression of CD40L by the CD40-expressing tumor cells may have profound effects on tumor cell growth,
differentiation, and apoptosis.
We did not have sufficient clinical information to correlate CD40L
expression in the CLL B cells with clinical features of autoimmune
disease in these patients. Given that the Fas antigen and its ligand
have been identified as critical mediators of apoptotic deletion of
peripheral, autoreactive B cells,60 we speculate that the
failure to delete autoreactive B cells in patients with CLL might be
due to failed Fas-mediated apoptosis in those nonmalignant cells.
Specifically, CD40L-stimulated B cells induced to express the Fas
antigen would be protected from Fas-mediated deletion by cross-linking
of the surface antigen receptor.26 Thus, most B cells that
receive inappropriate, CLL B-cell-derived help but bear Ig that is not
engaged by antigen would undergo apoptosis, which might account for the
hypogammaglobulinemia characteristic of this disease. However, B cells
responsive to a particular viral or other infectious agent present in
the host, or those reactive with autologous structures, might survive
Fas ligation subsequent to CD40 engagement and differentiate into
antibody-producing cells.
| |
FOOTNOTES |
Submitted October 2, 1997;
accepted January 23, 1998.
Supported with funds from The William H. Kearns Foundation, by The
Dorothy Rodbell Cohen Foundation for Sarcoma Research, and by National
Institutes of Health Grants No. R29CA71589 and P50 AR42588.
Address reprint requests to Elaine J. Schattner, MD, Room
C-640, Cornell University Medical College, 1300 York Ave, New York, NY
10021.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank the CLL patients and their physicians for providing
clinical samples. We thank Dr Radha K. Vakkalanka for expert guidance
in RT-PCR.
| |
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September 15, 1999;
163(6):
3116 - 3122.
[Abstract]
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Y. Zhang and E. Y. Denkers
Protective Role for Interleukin-5 during Chronic Toxoplasma gondii Infection
Infect. Immun.,
September 1, 1999;
67(9):
4383 - 4392.
[Abstract]
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F. Caligaris-Cappio and T. J. Hamblin
B-Cell Chronic Lymphocytic Leukemia: A Bird of a Different Feather
J. Clin. Oncol.,
January 1, 1999;
17(1):
399 - 399.
[Abstract]
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Abstract
Introduction
Abstract